The identification of amino acids present at the active site of enzymes and determination of their approximate arrangement in space is clearly important for understanding the basic facets of the mechanisms of enzyme catalyzed reactions. In particular, very little is known about the active site of pyruvate kinase, a branch point enzyme between the glycolytic and gluconeogenic pathways. Two approaches will be taken to study the active site of this enzyme: (1) synthesis of substrate analogs with nitroxide spin labels, using electron paramagnetic resonance (epr) techniques to study enzyme-substrate interactions; (2) synthesis of substrate analogs as active-site directed or active-site generated irreversible inhibitors.